SEMESTER OCTOBER-FEBRUARY 2024

LABORATORY REPORT
FOOD ANALYSIS (FST306)

EXPERIMENT 8
DETERMINATION OF CAFFEINE CONCENTRATION
BY USING HPLC

NAME STUDENT ID

INTAN BALQIS BINTI AHMAD 2023479936

MOHAMMAD FARIZAL BIN AZHAR 2023607932

MOHAMMAD RIDHWAN BIN AZMI 2023610602

MUHAMMAD AIMAN BIN MOHD REDZUAN 2023601988

PREPARED FOR :
DR. NOR MONICA BINTI AHMAD

DATE OF EXPERIMENT:
6 DECEMBER 2024
CONTENT

NO. TITLE PAGE

1. Introduction 2

2. Objectives 2

3. Materials and Apparatus 3

4. Procedure 3

5. Results 5

6. Calculation 6

7. Discussion 7-8

8. Conclusion 9

9. References 10

1
INTRODUCTION

Caffeine is a natural stimulant commonly found in coffee, tea, and various other beverages. It is
known for its stimulating effects on the central nervous system, which can enhance alertness and
reduce fatigue. High-Performance Liquid Chromatography (HPLC) is an advanced analytical
technique used in this experiment to separate, identify, and quantify components in a few
samples. HPLC is particularly effective for analyzing caffeine due to its high sensitivity,
precision, and accuracy.

Accurate measurement of caffeine levels is essential for quality control in the food and
beverage industry, ensuring that products meet the desired caffeine levels for consistency and
consumer satisfaction. Additionally, monitoring caffeine intake is important for individuals
sensitive to caffeine or those who need to manage their consumption for health reasons.
Regulatory compliance is another critical aspect, as there are regulations specifying maximum
allowable caffeine levels in certain products.

HPLC operates by passing a liquid sample through a column packed with a stationary
phase, where different components of the sample interact with the stationary phase to varying
degrees. This causes them to elute at different times, allowing for their separation and
quantification. In the industry, HPLC is widely used for quality control and assurance,
pharmaceutical analysis, environmental monitoring, and food and beverage testing. Its ability to
provide accurate and reliable results makes it an indispensable tool in various fields.

OBJECTIVES
1.​ To determine the retention time of the caffeine standard.
2.​ To identify the caffeine peak in soft drinks, Coke and Pepsi.
3.​ To quantify the concentration of caffeine presence in soft drinks, Coke and Pepsi.

2
MATERIALS AND APPARATUS

Standard
Caffeine standard, Acetonitrile, Deionized water

Sample
Soft drinks

Apparatus
Beaker, volumetric flask, dropper, syringe filter, vial

HPLC setting
HPLC model : 1200
Agilent Column : C18
Mobile phase : Acetonitrile : water (70:30)
Flow rate : 1.0 mL/min
Detector : UV at 254 nm
Injection volume : 20µL

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PROCEDURE

1.​ Caffeine standard solution

a.​ 10 mg of caffeine was weighed accurately into a 100 mL volumetric flask.
b.​ The caffeine then was dissolved in DI in water and made up to volume.
c.​ A 5 mL aliquot was filtered through a 0.45 µm pore diameter membrane filter to
remove particulate matter into the vial.

2.​ Sample preparation

a.​ About 10 mL of two different soft drinks sample was poured into a small beaker.
b.​ A 5 mL aliquot was filtered through a 0.45 µm pore diameter membrane filter to
remove particulate matter into the vial.
c.​ The solutions (standard & sample) were sonicated.

3.​ 10 µL of the caffeine standard solution was injected into the HPLC to determine the
retention time.
4.​ 10µL of the prepared soft drink sample was injected into the HPLC.
5.​ The peak area of caffeine was measured by referring to retention time.

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RESULTS

Table 1: Retention time and peak area of caffeine for standard and sample

Standard/sample Retention time, 𝑡𝑅 The peak area of caffeine

Caffeine standard 1.419 1235.74329

Coke 1.305 5609.75879

Pepsi 1.561 8124.47852

Table 2: Peak area data for caffeine and sample

Standard Peak area Response factor

Caffeine 1235.74329 12357.4329

Sample Peak area Amount

Coke 5609.75879 454.0734

Pepsi 8124.47852 657.6235

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CALCULATION

Caffeine’s concentration Amount

Concentration = 100 ppm 𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎
𝐴𝑚𝑜𝑢𝑛𝑡 = 𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑓𝑎𝑐𝑡𝑜𝑟

Caffeines’s Response Factor

𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 Amount of Coke
𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑓𝑎𝑐𝑡𝑜𝑟 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
5609.75879
= 12.3543
1235.74329
= 100
​
= 454.0734
=​ 12.3543

Amount of Pepsi
8124.47852
= 12.3543

= 657.6235

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DISCUSSION

In this experiment, High-Performance Liquid Chromatography (HPLC) was used to determine

the amount of caffeine in a few samples. This method is effective for analyzing the caffeine
because it is known for the component separation in the mixture. This experiment utilized
reverse phase HPLC, where the stationary phase is non-polar and the mobile phase is polar. The
stationary phase used for this experiment was C18 columns, where the polar compounds eluted
more quickly. As for the mobile phase, the setting used the 70:30 ratio of acetonitrile and water.
This ratio helps improve the elution of caffeine. This experiment then continued by determining
the retention time of the caffeine standard, followed by the sample used. This retention time is
known for the specific time caffeine takes to travel through the chromatographic column and
reach the detector. Next, the peak area for caffeine is determined for the caffeine standard and
samples. The peak area for caffeine is crucial for quantifying the concentration of caffeine in the
sample.

The caffeine standard solution was prepared by adding 10 mg of caffeine into a 100-mL
volumetric flask, followed by dissolving in distilled water. Then, the solution was filtered. This
method was applied to the samples used and continued by sonicating the solution. The retention
time obtained for the caffeine standard and the samples, Coke and Pepsi, was 1.419, 1.305, and
1.561, respectively. This then shows the peak area of caffeine for the solutions, which is
1235.74329, 5609.75879, and 8124.47852, respectively. Therefore, the peak area for Pepsi was
the highest, making it contain high concentrations of caffeine. By finding the response factor of
caffeine (12357.4329), the amount of caffeine in samples can be determined. In Coke, the
concentration of caffeine present was 454.0734. Unlike Pepsi, it contains a slightly higher
caffeine concentration, which is 675.6235. This finding can be supported by the fact that Pepsi
did have a higher caffeine content per 355 ml according to its nutritional labelling.

The differences in caffeine concentration in Coke and Pepsi might be due to the
formulation differences in both of them. It is known that caffeine content in Coke is 34 mg per
355 mL and in Pepsi is 38 mg per 355 mL. Pepsi may have slightly higher caffeine content as
part of its strategy to appeal to consumers seeking a stronger stimulant effect. Most of the

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reasons for the different values of caffeine are related to how the founders of these two
companies decided to pursue their customers. However, the peak area obtained for the caffeine
standard solution and the samples were not consistent. This might happen due to the mobile
phase composition, which is the ratio of acetonitrile and water that was not suitable for the
sample used. In reversed-phase HPLC, increasing the proportion of polar solvents enhances
interactions with polar analytes, leading to longer retention times and potentially better
separation. Other than that, it might be due to how the injection process was done. This is very
sensitive due to its small amount since 1 µL can lead to a 10% error in the results.

8
CONCLUSION

In conclusion, the experiment on the determination of caffeine concentration by using

High-Performance Liquid Chromatography (HPLC) was managed to determine the retention
time of the caffeine standard, identify the caffeine peak in soft drinks, and quantify the caffeine
content in these beverages. The retention times were identified as 1.419 minutes for the caffeine
standard, 1.305 minutes for Coke, and 1.561 minutes for Pepsi. The concentrations of caffeine
were found to be 0.4540 mg/mL in Coke and 0.6575 mg/mL in Pepsi, mentioning that Pepsi has
a higher caffeine content. However, the experiment faced limitations such as inconsistent peak
areas, possibly due to the mobile phase composition and injection process errors. In the future,
through careful calibration and optimization of experimental conditions, more accurate and
reliable caffeine concentrations can be determined. This study highlights how crucial HPLC is
for precisely determining caffeine levels, which is helpful for both quality control in the beverage
industry and for consumers keeping an eye on their intake.

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REFERENCES

Aljofan, M., Alkhamaisah, S. I., Younes, K. M., & Gaipov, A. (2019). Development and

validation of a simple and sensitive HPLC method for the determination of liquid form of

therapeutic substances. Electronic Journal of General Medicine, 16(6), em166.

https://doi.org/10.29333/ejgm/112271

Caffeine Content for some of the Most Popular Drinks Revealed. (n.d.). Www.vidrate.com.

https://www.vidrate.com/blogs/news/caffeine-content-for-some-of-the-most-popular-drin

ks-revealed

HPLC Basics, US. (n.d.). Www.thermofisher.com.

https://www.thermofisher.com/my/en/home/industrial/chromatography/chromatography-l

earning-center/liquid-chromatography-information/hplc-basics.html

Pokhrel, P., Shrestha, S., Rijal, S. K., & Rai, K. P. (2016). A simple HPLC Method for the

Determination of Caffeine Content in Tea and Coffee. Journal of Food Science and

Technology Nepal, 9, 74–78. https://doi.org/10.3126/jfstn.v9i0.16200

Tom. (2023, June 20). How much caffeine is in Pepsi? (Compared to coffee). Batch Coffee Club

UK. https://www.batchcoffee.co.uk/stories/how-much-caffeine-in-pepsi

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